Neddylation is an essential posttranslational modification, and a goal of my research program is to define how posttranslational modifications can be manipulated to regulate T cell receptor (TCR) complex signaling, T-cell activation, and effector functions to ultimately treat immune-mediated disease. An important unanswered question is how neddylation regulates T-cell function. The studies in this R21 proposal will explore and provide foundational data in this poorly understood area. Prior to our preliminary studies, it was unknown how neddylation regulates T-cell receptor (TCR) complex signaling and T cell function. We used, a highly selective NEDD8-activating enzyme (NAE1) inhibitor that blocks all protein neddylation, as a tool to determine if inhibition of neddylation impacts T-cell function. We found that inhibition of neddylation leads to increased IL-2 production, which serves as a biological readout for TCR signal strength, as well as enhanced proliferation, increased activation marker expression, and enhanced Tregs development in vitro. In addition, we treated SKG mice, which develop inflammatory arthritis due to a known defect in TCR complex signaling, with the neddylation inhibitor and observed reduced arthritis progression and increased numbers of IL-2 producing T cells in vivo. Collectively, these studies suggest that the neddylation inhibitor, a drug already being used in phase-I clinical trials for patients with malignancies, enhances T-cell function and may be a treatment option for autoimmune disease. Thus we hypothesize that defining proteins that are neddylated in T cells and regulate TCR complex signaling will lead to greater insights into ways to manipulate T cell-function to treat immune mediated disease. To begin to fill the gap in our understanding of how neddylation regulates TCR complex signaling, we will take an unbiased approach and test our hypothesis through the following specific aims: Aim 1. To identify proteins that are neddylated upon TCR complex signaling, we will perform difference in-gel electrophoresis (DIGE) and mass spectrometry. Aim 2. To determine if the proteins that are inducibly neddylated upon TCR complex signaling inhibit TCR signaling, we will knockdown expression of the protein and generate T cell lines that express a non-neddylatable mutant of the protein. We will quantitate IL-2 production and activation marker induction as a biological readout for TCR signal strength. To achieve these goals, we have generated the necessary reagents and have assembled a collaborative team with proteomic expertise to perform the proposed studies. At the conclusion of these studies, we will have expanded our knowledge of the regulation of TCR complex signaling by identifying proteins that are inducibly neddylated and validating their importance in regulating TCR complex signaling and T- cell effector functions. Defining how neddylation regulates TCR complex signaling should lead to new opportunities to specifically manipulate T-cell function in a variety of immune mediated diseases.